Herpes simplex virus (HSV) is the leading cause of blindness due to infectious disease in the United States, and affects approximately 500,000 people a year. Different strains of virus have distinctive virulence phenotypes, indicating that specific determinants in the virus affect disease severity. Virulence is a multigenic phenomenon and likely requires interactions between viral proteins and viral and host proteins. We and others have previously demonstrated that mixed infections with two HSV strains can cause more severe disease due to complementation or recombination. These results imply that at least two virulence determinants are involved in mixed infections and the identification of the determinants provides a way to identify components cooperating in virulence. We have previously identified a pair of HSV-1 strains, OD4 and CJ394, that caused more severe ocular disease and neurovirulence when mixed. Available evidence indicates OD4 and CJ394 are not deletion mutants, suggesting different alleles may be involved in the increased virulence seen when the viruses are mixed. RFLP mapping of the genomes of 4 virulent OD4/CJ394 recombinants suggested that the EcoR1 H fragment of OD4 might carry a virulence determinant. We have now shown that the cloned OD4 EcoR1 H fragment (US1 to US12 genes) can indeed transfer increased virulence to CJ394. The major goals of this proposal are to (1) use subclones of the OD4 EcoRI H fragment in marker transfer to map the regions involved, (2) to sequence the respective regions of both OD4 and CJ394, and (3) to identify the amino acids involved in the virulence differences using site directed mutagenesis. Information on the role of multiple virulence determinants and their interactions will be critical for improving prevention and treatment of ocular disease, the use of HSV based vectors for gene delivery, and attenuated HSV vaccines.